The relative contribution of de novo and salvage nucleotide pools is an important parameter in the rational design of antipyrimidine therapies, but was not previously measured in vivo because of the lack of suitable methodologies. Using stable isotopes, GC/MS- methodology, and a novel method of data analysis, we measured de novo synthesis in mouse tumors and tissues. These values were compared with those for salvage determined with radiolabelled uridine. In a panel of mouse tumors, the contribution of de novo synthesis was at least four times the contribution of salvage synthesis. For normal tissues; de novo was more important in intestine, salvage more important in kidney, and the liver was dependent equally on de novo and salvage. Previous studies by our group demonstrated that an excess of ammonia can increase pyrimidine synthesis in liver and intestine because of the presence of mitochondrial carbamyl phosphate synthetase I (an enzyme lacking in most tumors). Host toxicity to PALA was reduced by providing excess ammonia by increasing dietary protein. Thus, it may be possible to improve the selectivity and therapeutic effectiveness of antipyrimidines through diet manipulation. Murine tumors made resistant to 5-fluorouridine were cross-resistant to cyclopentenylcytosine. The mechanism is related to the over-production of CTP, possibly through a change in CTP-synthase. Studies on the cellular pharmacology of DUP-785 showed that there is a direct correlation between inhibition of de novo pyrimidine synthesis, changes in pyrimidine nucleotide concentrations, and cell proliferation following short (<24 h) drug exposures; however, with prolonged exposures (>24 h), there is a departure from this correlation in that restoration of pyrimidine nucleotide pools and de novo pathway activity does not restore cell proliferation. This information should be useful to the design of clinical protocols with this agent.